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. 2026 Jun 15;68(11):3914–3928. doi: 10.1111/jipb.70322

Natural variation in the promoter of OsPDX1.2 enhances vitamin B6 accumulation and cold tolerance in rice

Bi Wang 1, Qiu Yi Wang 1, Wen Jie Shi 1, Rong Hui Li 1, Xu Dong Zhang 1, Li Ping Wang 1, Shi Jia Xia 1, Kai Xi Chen 1, Jun Jie Zhou 2, Yang Yang Sun 3,4, Jing He 1, Alisdair R Fernie 5, Ying Chun Han 6, Jun Zhou Li 6, Yu Heng Shi 2, Xian Qing Liu 1, Yuan Yuan Zhang 1,✉, Jie Luo 1,2,✉, Cheng Jin 1,2,✉
PMCID: PMC13725607  PMID: 42290547

ABSTRACT

Vitamin B6 (VB6) is an essential nutrient that must be obtained from the diet by humans and livestock, and plays an important role in response to various biotic and abiotic stresses in plants. Although the VB6 biosynthesis pathway has been uncovered in higher plants, understanding of its genetic control remains limited, and the role of VB6 in responding to cold stress is largely unknown. Here, we showed that variation in VB6 levels in rice is attributable to differential transcription of OsPDX1.2, a pyridoxal phosphate synthase, due to polymorphisms in its promoter. Natural variations in the OsPDX1.2 promoter affect the binding affinity of OsbZIP18, a transcriptional factor repressing OsPDX1.2 transcription. Loss of function in OsPDX1.2 or overexpression of OsbZIP18 significantly reduces cold tolerance in rice. Further investigation revealed that both genes affect cold tolerance by regulating VB6 and abscisic acid (ABA) biosynthesis. Moreover, the frequency of the cold‐tolerant pOsPDX1.2 Hap2 increases at higher latitudes, whereas the cold‐sensitive pOsPDX1.2 Hap1 increases at lower latitudes, indicating that natural variation in OsPDX1.2 contributes to differential cold adaptation in rice. Overall, our results reveal a regulatory pathway controlling rice VB6 accumulation and cold tolerance via the OsbZIP18–OsPDX1.2 module, providing a potential target for improving rice quality and stress tolerance.

Keywords: ABA, cold tolerance, natural variation, stress tolerance, vitamin B6

Allelic variation in the promoter of the pyridoxal phosphate synthase OsPDX1.2 caused variation in the ACE‐box cis‐elements, resulting in differing OsPDX1.2 expression due to differences in binding affinity between the OsbZIP18 transcription factor and the ACE‐box, contributing to variation in vitamin B6 levels and cold tolerance among rice varieties.

graphic file with name JIPB-68-3914-g004.webp

INTRODUCTION

Vitamin B6 (VB6), a water‐soluble vitamin essential for all living organisms, consists of six related compounds: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated forms pyridoxine 5′‐phosphate (PNP), pyridoxal 5′‐phosphate (PLP), and pyridoxamine 5′‐phosphate (PMP) (Fitzpatrick et al., 2007; Roje, 2007). PLP acts as a cofactor involved in catalyzing over 140 biochemical reactions, predominantly in amino acids, glucose, and lipid metabolism, surpassing any other known coenzyme in reaction diversity (Percudani and Peracchi, 2003; Mooney and Hellmann, 2010; Colinas et al., 2016). Vitamin B6 cannot be synthesized by humans or animals, and must therefore be acquired from their diet, primarily from plant‐based foods (Fitzpatrick et al., 2012). To date, our understanding of VB6 metabolic regulation in plants remains limited, and yet, it is crucial for biofortification efforts.

In plants, the de novo VB6 biosynthesis pathway involves two cytosolic enzymes: PDX1 (pyridoxal phosphate synthase) and PDX2 (Burns et al., 2005). PDX2 catalyzes the release of ammonia from glutamine, which PDX1 subsequently combines with ribose 5‐phosphate and glyceraldehyde 3‐phosphate to synthesize PLP (Tambasco‐Studart et al., 2005, 2007). The rice genome contains three PDX1 homologs (OsPDX1.1, OsPDX1.2, and OsPDX1.3) (Chen et al., 2014; Liu et al., 2022b). Total VB6 levels decreased in ospdx1.2 knockout plants, but PN and its derivative increased in OsPDX1.1‐overexpressing plants, indicating both genes' essential roles in VB6 biosynthesis (Chen et al., 2018; Liu et al., 2022b). However, the biochemical functions of these genes (including OsPDX1.1, OsPDX1.2, and OsPDX1.3) remain uncharacterized in rice. Moreover, to our knowledge, no transcription factors regulating VB6 biosynthesis have been identified in plants.

VB6 functions as a potent reactive oxygen species (ROS)‐scavenging antioxidant and plays a key role in biotic and abiotic stress responses in plants (Danon et al., 2004; Havaux et al., 2009; Leasure et al., 2011; Leuendorf et al., 2014; Moccand et al., 2014; Dell'Aglio et al., 2017; Czegeny et al., 2019; Liu et al., 2022b, 2022c, 2025a). For example, the exogenous application of PN can significantly alleviate salt toxicity symptoms and reduce ROS accumulation in rice (Nan et al., 2020). Recent research showed that the effector protein AvrRxo1 enhances Xanthomonas oryzae pv. oryzicola (Xoc) virulence and suppresses stomatal immunity by targeting and subsequently degrading OsPDX1 (Liu et al., 2022b). In Arabidopsis, AtPDX1 expression is induced by abiotic stresses such as drought, high light, ozone stress, and UV radiation, whereas the Atpdx1.3 mutant shows hypersensitivity to salt, high light, photo‐oxidative, and UV‐B stress (Titiz et al., 2006; Wagner et al., 2006; Denslow et al., 2007; Dell'Aglio et al., 2017). Cold stress is a major abiotic stress that adversely affects plant growth and crop productivity. Previous studies indicate that cold stress induces protective proteins and metabolites (e.g., compatible solutes and ROS scavengers) to enhance tolerance (Kaplan et al., 2004; Guy et al., 2007; Zhu et al., 2007; Maruyama et al., 2014; Zhang et al., 2016). Nevertheless, the role of VB6 in cold stress response and its molecular regulatory mechanisms in plants remain largely unknown.

Previous studies demonstrate substantial genetic variation in VB6 accumulation in different tissues among rice cultivars (Chen et al., 2014; Mangel et al., 2022). By using multi‐connected biparental chromosome segment substitution line populations, four quantitative trait loci associated with the accumulation of PN and its derivative have been identified in flag leaves (Chen et al., 2018). However, the natural allelic variation underlying VB6 accumulation remains unknown and requires further investigation.

In this study, we identified OsPDX1.2 as a major determinant of rice VB6 content by combining a metabolite‐based genome‐wide association study (mGWAS) with functional analysis. A natural variant in the OsPDX1.2 promoter affects its expression by altering binding to the bZIP transcription factor OsbZIP18. Further study revealed that OsPDX1.2 positively regulates cold tolerance by promoting the synthesis of VB6 and abscisic acid (ABA). Geographical association analysis revealed that the natural variation of OsPDX1.2 is associated with divergence in cold adaptation among rice varieties.

RESULTS

Genetic basis of natural variation in VB6 levels revealed by mGWAS

Using high‐throughput LC‐MS/MS analysis, we identified a total of seven VB6 vitamers and derivatives in the leaves of ZH11 (var. japonica, cv Zhonghua 11), namely, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′‐phosphate (PLP), pyridoxine O‐glucoside (PN‐G), 4‐pyridoxic acid (4PA), and 4‐pyridoxic acid O‐glucoside (4PA‐G) (Table S1). The signal intensity of PLP, the biologically active coenzyme form of VB6, was lower than PN and its derivatives (Figure S1). Subsequently, we analyzed the natural variation of PN and its derivatives PN‐G, 4PA, and 4PA‐G in a core collection of 512 rice accessions, comprising 290 indica, 151 japonica, and 71 other rice cultivars (Figure S2A; Dataset S1). The results revealed substantial variations in the contents of PN, PN‐G, 4PA, and 4PA‐G among the rice cultivars, with coefficients of variation (CV) ranging from 58% to 101% (Figures 1A, S2B–E). A strong positive correlation was observed between PN and PN‐G, 4PA, and 4PA‐G, as well as PN‐G and 4PA‐G (Figure 1B). Among these subpopulations, the japonica cultivars demonstrated the highest accumulation of all four vitamers (PN, PN‐G, 4PA, and 4PA‐G), while indica cultivars displayed a wide range of variation in the contents of PN and PN‐G (Figures 1C–F, S2F–I).

Figure 1.

Figure 1

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Natural variation of vitamin B6 (VB6) components in rice leaves

(A) Heatmap of the natural variation and coefficient of variation (CV) of VB6 vitamers in rice. The content value of each VB6 is normalized and hierarchical clustering is performed. The color bar indicates Z‐score values, with red and blue representing relatively higher and lower values, respectively. PN, Pyridoxine. PN‐G, pyridoxine O‐glucoside. 4PA, 4‐pyridoxic acid. 4PA‐G, 4‐pyridoxic acid O‐glucoside. (B) Correlations among PN, PN‐G, 4PA, and 4PA‐G. The color bar indicates Pearson s correlation coefficients (r). (C–F) Frequency distribution of the contents of PN (C), PN‐G (D), 4PA (E), and 4PA‐G (F) in 512 rice accessions. (G–H) Manhattan plots of GWAS and quantile–quantile (QQ) plots of PN (G) and PN‐G (H) in the full population. (I, J) Manhattan plots of GWAS and QQ plots of PN (I) and PN‐G (J) in the indica subpopulation.

Subsequently, mGWAS were conducted using a mixed linear model (MLM) in the full population, as well as in the indica and japonica subpopulations. Applying Bonferroni‐corrected genome‐wide significance thresholds (PLMM = 6.6 × 10−8 for the full population, 8.7 × 10−8 for indica, and 2.0 × 10−7 for japonica), three major loci located on chromosomes 7, 9, and 10 were detected for both PN and PN‐G (Figure 1G–J; Table S2). Notably, these associations were consistently identified in both the full population and the indica subpopulation (Figure 1G–J). The phenotypic variation explained by each locus ranged from 6.52% to 10.38%, with the locus located on chromosome 10 demonstrating the strongest effects for both PN and PN‐G in the full population (Table S2).

Natural variations in OsPDX1.2 promoters predominantly determine VB6 levels in rice

We further focused our attention on the significant association signal in a 48‐kb genomic interval block (0.044–0.092 Mb) on chromosome 10, which contained five protein‐coding genes, as annotated in the Nipponbare reference genome (Figures 2A, S3A). To ascertain the causal gene underlying this locus, we performed a transcriptome‐wide association study (TWAS) using leaf transcriptome data of 287 rice accessions obtained from a previous study (Liu et al., 2022a). This analysis refined the candidate set from the initial five genes to a single candidate, LOC_Os10g01080 (OsPDX1.2), a pyridoxine synthase belonging to the SOR/SNZ family, whose expression level showed a positive correlation with both PN and PN‐G accumulation (Figures 2B–E, S3B–E).

Figure 2.

Figure 2

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Natural variations in OsPDX1 .2 are associated with VB6 accumulation in rice

(A) Local Manhattan plots of GWAS results of PN and PN‐G in the indica subpopulation. The corresponding linkage disequilibrium (LD) heatmap for the candidate region (demarcated by red dashed lines) is shown below. The candidate interval encompassing the peak is indicated by red vertical lines, while red dots represent SNPs within the promoter region of OsPDX1.2; these corresponding SNPs are also highlighted with red lines in the LD heatmap. (B, C) Local transcriptome‐wide association study (TWAS) for PN (B) and PN‐G (C). Red dots indicate association signals corresponding to OsPDX1.2. (D, E) Correlation between OsPDX1.2 expression and levels of PN (D) and PN‐G (E) in 161 indica accessions. The r values were derived from Pearson correlation analysis. (F) Haplotype analysis of the polymorphic promoter region of OsPDX1.2 in the indica subpopulation. (G–I) Comparative analysis of PN (G), PN‐G (H), and OsPDX1.2 expression level (I) between two distinct haplotypes. P values were calculated using the Wilcoxon rank‐sum test. (J, K) Comparison of promoter activity between different haplotypes of OsPDX1.2. The relative luciferase activities measured by the dual‐luciferase reporter assay system. Data are shown as the means ± SE (n = 3). Statistical significance was determined by two‐sided Student's t‐tests.

To investigate the contribution of natural variation in OsPDX1.2 to the variations of PN and PN‐G levels in rice leaves, we conducted a haplotype analysis of OsPDX1.2 in both the full population and the indica subpopulation. Seven single‐nucleotide polymorphisms (SNPs) were identified in the promoter region of OsPDX1.2, whereas no nucleotide substitutions were detected within its coding sequence (CDS) (Figure S3A; Table S3). We therefore classified the full population into four haplotypes according to the variation in the OsPDX1.2 promoter region (Figure S3F). Notably, the indica subspecies contains two major haplotypes (pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 ), whereas pOsPDX1.2 Hap1 is absent in japonica rice (Figures 2F, S3F). The rice accessions carrying the pOsPDX1.2 Hap1 allele showed significantly lower levels of PN, PN‐G, and OsPDX1.2 expression than pOsPDX1.2 Hap2 (Figures 2G–I, S3G–I). Transient expression assays in Nicotiana benthamiana leaves revealed that the promoter segments from pOsPDX1.2 Hap2 showed 6.2‐fold higher activity than those from pOsPDX1.2 Hap1 (Figure 2J, K). These results provide support for the impact of natural OsPDX1.2 promoter variations on the transcription of this gene.

OsPDX1.2 positively regulates the levels of VB6

The expression pattern of OsPDX1.2 at different growth stages was next investigated with real‐time quantitative PCR (RT‐qPCR), and the results showed that OsPDX1.2 is predominantly expressed in leaves during the vegetative growth stage (Figure 3A). Subcellular localization assays using transient expression of 35S:eGFP–OsPDX1.2 in N. benthamiana leaves revealed that the fusion protein localizes predominantly to the cytoplasm (Figure 3B), which was further confirmed by similar results obtained in rice protoplasts (Figure S4). In vitro assays demonstrated that the OsPDX1.2 enzyme catalyzes the direct synthesis of PLP from ribose 5‐phosphate (R5P), glyceraldehyde 3‐phosphate (G3P), and glutamine (Gln), thereby confirming the role of OsPDX1.2 as a functional PLP synthase (Figure 3C, D). To further investigate the function of OsPDX1.2 in VB6 metabolism, we generated ospdx1.2 knockout mutants (ospdx1.2‐1, ospdx1.2‐2, and ospdx1.2‐3) using the CRISPR/Cas9 system (Figure S5A). Metabolite profiling revealed a significant reduction in the accumulation of VB6 vitamers in the leaves and seeds of ospdx1.2 mutants compared to the wild type (Figures 3E–I, S5B–F). Conversely, OsPDX1.2‐overexpressing plants showed significantly increased accumulation of VB6 vitamers in leaves compared to the wild type (Figure S6). These results collectively indicate that OsPDX1.2 encodes a functional PLP synthase and positively regulates VB6 biosynthesis in rice.

Figure 3.

Figure 3

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Functional characterization of OsPDX1.2 in rice

(A) Relative expression levels of OsPDX1.2 in different tissues at various growth stages. (B) Subcellular localization of OsPDX1.2. CBL1‐OFP was used as a plasma membrane marker. Scale bar = 20 μm. (C) Schematic of VB6 biosynthesis and metabolism in plants. (D) In vitro enzyme activity assays of OsPDX1.2 protein. (E–I) Relative content of PLP (E), PL (F), PM (G), PN (H), and PN‐G (I) in leaves of wild‐type and ospdx1.2 mutant plants. Data are shown as the means ± SE (n = 3). Statistical significance was determined by one‐way ANOVA (Tukey's test).

OsPDX1.2 positively regulates cold tolerance in rice seedlings

Previous studies have shown that the expression of PDX1 genes is induced by various abiotic stresses, including high light, drought, and UV radiation in plants (Denslow et al., 2007; Titiz et al., 2006). The expression of OsPDX1.2 was significantly upregulated by cold stress (Figure S7), suggesting that it may also play an important role in the rice cold stress response. To test this hypothesis, we analyzed the phenotype of the ospdx1.2 mutant under cold stress. Under normal growth conditions, no obvious phenotypic differences were observed between ospdx1.2 mutants and the wild type (Figure 4A). However, after 5 d of cold stress treatment (4°C), both ospdx1.2 mutants were more susceptible to cold stress, displaying significantly lower biomass and survival rates than the wild type after a 7‐d recovery at 25°C (Figure 4C–E). Consistent with this observation, histochemical staining using 3,3‐diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) showed enhanced reactive oxygen species (ROS) accumulation in the leaves of ospdx1.2 mutants after cold stress (Figure 4F). The levels of malondialdehyde (MDA) in ospdx1.2 mutants were significantly increased under cold stress (Figure 4B). Conversely, OsPDX1.2‐overexpressing plants showed enhanced cold tolerance, with higher biomass, survival rate, and reduced ROS accumulation and MDA levels compared to the wild type under cold stress (Figure S8). Furthermore, exogenous application of PN significantly rescued the cold sensitivity of rice seedlings (Figure 4), suggesting that OsPDX1.2 positively regulates cold tolerance by regulating VB6 synthesis at the seedling stage.

Figure 4.

Figure 4

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Exogenous VB6 and ABA application rescues the cold‐sensitive phenotype of the ospdx1.2 mutant

(A) Phenotypic analysis of cold tolerance in wild‐type and ospdx1.2 mutant plants with exogenous VB6 and ABA application. Twelve‐d‐old plants grown under normal conditions were sprayed daily with 30 μM PN (VB6), 30 μM ABA, or distilled water (control) during the 5‐d treatment at 4°C, followed by a 7‐d recovery at 25°C. Scale bar, 5 cm. (B) MDA contents in leaves of 12‐d‐old ospdx1.2 transgenic and wild‐type rice seedlings after a 24‐h treatment at 4°C. (C–E) Fresh weight (C), fresh weight increase (D), and survival rate (E) of plants measured after recovery from the 4°C treatment. (F) Histochemical analysis of reactive oxygen species (ROS) in leaves under cold stress. Hydrogen peroxide (H2O2) and superoxide anion (O2 −) accumulations were detected using diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. Seedlings were treated at 4°C for 24 h prior to staining. Scale bar, 1 cm. In (B, E), data are presented as means ± SE (n = 3). In (C, D), data are presented as means ± SE (n = 16). Statistical significance for panels (B–E) was determined by one‐way ANOVA (Tukey's test).

Recent studies indicate that VB6 is required as a cofactor in the biosynthesis of abscisic acid (ABA), which positively regulates the cold stress response in plants (Ji et al., 2011; Liu et al., 2022b; Li et al., 2025b). This occurs because VB6 acts as an ABA3 (molybdenum cofactor sulfurase) cofactor that converts molybdenum cofactor into its sulfurated form, which is in turn essential for aldehyde oxidase AAO3 to catalyze the conversion of abscisic aldehyde into ABA (Seo et al., 2000, 2001; Bittner et al., 2001). A significant decrease in ABA accumulation was detected in the leaves of ospdx1.2 mutants (Figure S9A), suggesting that the cold stress‐sensitive phenotype was likely attributable to an ABA deficiency. In contrast, the ABA content was significantly increased in OsPDX1.2‐overexpressing plants compared to the wild type (Figure S9B). Exogenous ABA complemented the cold‐sensitive phenotype of the ospdx1.2 mutants (Figure 4). These results indicate that OsPDX1.2 potentially confers cold tolerance probably via the regulation of both VB6 and ABA biosynthesis.

Natural variation in OsPDX1.2 contributes to adaptation to cold stress in rice

Given that OsPDX1.2 can regulate cold tolerance in the ZH11 cultivar, we explored whether its natural variation is associated with cold stress response in rice populations. A subset of 36 randomly selected accessions (18 pOsPDX1.2 Hap1 and 18 pOsPDX1.2 Hap2 ) from the indica subpopulation was evaluated for cold tolerance to minimize confounding effects from genetic background variation across subpopulations. The results showed that pOsPDX1.2 Hap2 plants had enhanced cold tolerance compared to pOsPDX1.2 Hap1 plants, accompanied by increased PN and PN‐G contents, elevated OsPDX1.2 expression, and improved survival rates under cold stress (Figures 5A–D, S10). Furthermore, we found that OsPDX1.2 showed a strong association with latitude, with pOsPDX1.2 Hap2 occurring in higher latitudes, while pOsPDX1.2 Hap1 was more frequently distributed in lower latitudes (Figure 5E–G; Dataset S2). These data suggest that pOsPDX1.2 Hap2 may facilitate adaptation to cold environments in rice.

Figure 5.

Figure 5

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Natural variation of OsPDX1.2 is associated with cold tolerance and latitudinal adaptation in rice

(A–C) Comparative analysis of PN (A), PN‐G (B), and OsPDX1.2 transcript levels (C) between the pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 haplotypes in 36 indica accessions. (D) Survival rates of pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 under cold stress in the indica rice population. Eight‐d‐old seedlings were exposed to 4°C for 4 d and then recovered at 25°C for 7 d. (E) Global distribution of 709 cultivated rice accessions carrying the pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 alleles. (F) Latitude distribution of pOsPDX1.2 haplotypes in 709 rice accessions. P values were calculated using the Wilcoxon rank‐sum test. (G) The line chart depicts the relationship between allele frequency and latitude for the pOsPDX1.2 haplotypes. In (A–D), data are presented as means ± SE (n = 18). Statistical significance was determined by two‐sided Student's t‐tests.

OsbZIP18 negatively regulates cold tolerance in rice

Since natural variation in the OsPDX1.2 promoter contributes to the difference in OsPDX1.2 gene expression and VB6 accumulation among rice varieties (Figure 2D–K), we compared the cis‐acting elements in pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 promoter regions. The results showed that a guanine (G) to cytosine (C) point mutation at the −559 position leads to the gain of an ACE cis‐element (ACGT) (Figure 6A), a well‐characterized canonical binding site for bZIP transcription factors (Gangappa and Botto, 2016; Dröge‐Laser and Weiste, 2018; Dröge‐Laser et al., 2018). Therefore, we performed a co‐expression analysis using the public database ATTED‐II (https://atted.jp/top_search/#CoExSearch), which identified OsbZIP18 as a candidate (Table S4).

Figure 6.

Figure 6

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Exogenous applications of VB6 and ABA enhance cold stress tolerance in OsbZIP18 ‐overexpressing rice plants

(A) Diagram of the pOsPDX1.2 Hap1 and pOsPDX1.2 Hap2 regions. Pink rectangles indicate ACE cis‐elements, known binding sites for bZIP transcription factors. The SNP (vg1000082732) that distinguishes the two haplotypes is highlighted in red. (B, C) Vitamin B6 (B) and ABA (C) contents in leaves of wild‐type and OsbZIP18‐overexpressing (OE) plants. (D) Phenotypic analysis of cold tolerance in wild‐type and OsbZIP18_OE plants following exogenous VB6 and ABA supplementation. Twelve‐d‐old seedlings grown under standard conditions received daily foliar sprays of 30 μM PN (VB6), 30 μM ABA, or distilled water (control) throughout the 5‐d cold treatment at 4°C, after which plants were transferred to 25°C for a 7‐d recovery period. Scale bar, 5 cm. (E) MDA contents in leaves of 12‐d‐old OsbZIP18_OE transgenic and wild‐type seedlings after a 24‐h treatment at 4°C. (F–H) Fresh weight (F), fresh weight increase (G), and survival rate (H) of plants measured after recovery from cold stress. (I) Accumulation of reactive oxygen species in cold‐stressed leaves, visualized by DAB (for H2O2) and NBT (for O2 −) staining. Scale bar, 1 cm. In (E, H), data are presented as means ± SE (n = 3). In (F, G), data are presented as means ± SE (n = 24). Statistical significance was determined by one‐way ANOVA (Tukey's test).

To determine whether OsbZIP18 is involved in regulating VB6 accumulation and the cold stress response, we generated OsbZIP18‐overexpressing transgenic rice lines. Consistently, OsbZIP18_OE plants displayed markedly reduced levels of VB6 vitamers, ABA as well as diminished cold tolerance compared to the wild type (Figure 6B, C). Conversely, no significant differences in OsPDX1.2 expression, VB6 levels, or ABA content were detected between the osbzip18 mutant and the wild type (Figure S11), which may be explained by functional redundancy between OsbZIP18 and OsbZIP48 (Burman et al., 2018; Xiong et al., 2024). Moreover, the application of VB6 and ABA dramatically significantly alleviated the cold sensitivity of the OsbZIP18_OE plants mutants (Figure 6D–I). These results demonstrate that OsbZIP18 regulates cold tolerance by modulating the biosynthesis of VB6 and ABA.

Natural variations in the OsPDX1.2 promoter affect the binding of OsbZIP18

To investigate whether OsPDX1.2 is a target of OsbZIP18, we first analyzed the expression level of OsPDX1.2 in OsbZIP18_OE plants. Indeed, OsPDX1.2 expression was significantly downregulated in OsbZIP18_OE plants (Figure 7A). Yeast one‐hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) confirmed the ability of OsbZIP18 to bind the ACE motif (Figure 7B, C). These results indicate that OsbZIP18 acts as a negative regulator of OsPDX1.2.

Figure 7.

Figure 7

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The OsbZIP18–OsPDX1.2 regulatory module underlies natural variation in rice cold tolerance

(A) Transcript levels of OsPDX1.2 in leaves of OsbZIP18_OE plants. The relative expression of OsPDX1.2 was measured by RT–qPCR normalized to Ubiquitin. Data are shown as the means ± SE (n = 3). Statistical significance was determined by one‐way ANOVA (Tukey's test). (B) Yeast one‐hybrid assay demonstrating the binding of OsbZIP18 to the OsPDX1.2 promoter. The combination of empty pGADT7 and pHIS2‐OsPDX1.2 was co‐transformed as a negative control. (C) Electrophoretic mobility shift assay (EMSA) of OsbZIP18 binding to the ACE motif in the OsPDX1.2 promoter. Competitive binding assays were performed using five‐ and 20‐fold molar excess of unlabeled probe. (D) Diagram of the site‐directed mutagenesis locations in the OsPDX1.2 promoter region. (E) Schematic representation of the effector and reporter constructs for transient expression in N. benthamiana leaf epidermis. (F, G) Transcriptional repression of pOsPDX1.2 C and pOsPDX1.2 G by OsbZIP18. Data are shown as the means ± SE (n = 4). Statistical significance was determined by two‐sided Student's t‐tests. (H) A proposed model for natural polymorphism in OsPDX1.2 regulating cold tolerance through vitamin B6. The transcription factor OsbZIP18 represses OsPDX1.2 expression and vitamin B6 accumulation. Natural variation in the promoter ACE element modulates this regulation: Stronger OsbZIP18 binding to the pOsPDX1.2 Hap1 allele more strongly suppresses OsPDX1.2, thereby altering ROS and ABA homeostasis and resulting in enhanced cold sensitivity in pOsPDX1.2 Hap1 lines compared to pOsPDX1.2 Hap2 lines. Arrows and T‐bars denote promotion and inhibition, respectively.

Subsequently, dual‐luciferase assays were conducted to determine the transactivation ability of OsbZIP18 with respect to the OsPDX1.2 promoters. A G‐to‐C point mutation was introduced at the −559 bp position of the OsPDX1.2 promoter in the background of Nipponbare (pOsPDX1.2 Hap2 ) via site‐directed mutagenesis, generating the pOsPDX1.2 C allele, while the original allele was designated as pOsPDX1.2 G (Figure 7D). After co‐transforming 35S:OsbZIP18 with the pOsPDX1.2 G or pOsPDX1.2 C constructs in N. benthamiana leaves, the activity of both promoters was suppressed, as compared with that of the vector control (Figure 7E–G). Moreover, the luciferase activity driven by the pOsPDX1.2 C promoter was substantially lower than that of the pOsPDX1.2 G promoter (Figure 7F, G), indicating that OsbZIP18 represses the activity of the OsPDX1.2 promoter, especially that of pOsPDX1.2 C .

DISCUSSION

Improving quality and stress tolerance is a key goal in crop breeding particularly now, given the dramatic challenges imposed by climate change (Tao et al., 2025). VB6 is an essential nutrient for humans and plays a crucial role in mediating tolerance to both biotic and abiotic stresses in plants (Fitzpatrick et al., 2012; Fudge et al., 2017; Liu et al., 2022d; Fitzpatrick, 2024). Understanding the molecular mechanisms underlying VB6 accumulation and its genetic bases is crucial for developing both VB6‐rich and stress‐tolerant crop varieties. In this study, we identified OsPDX1.2 as a key regulator of VB6 accumulation and cold tolerance in rice. We discovered that sequence variation at SNP‐559 in the OsPDX1.2 promoter determines the binding affinity of OsbZIP18 and is responsible for the diverse OsPDX1.2 expression levels and cold tolerance variation in rice population. This study uncovers a novel regulatory module governing VB6 accumulation and cold tolerance in rice (Figure 7H).

We provide a series of evidence demonstrating that OsPDX1.2 in rice (i) is essential for VB6 synthesis and cold tolerance; (ii) regulates cold tolerance by mediating VB6 and ABA accumulation; and (iii) is transcriptionally regulated by OsbZIP18, which is critical for cold responses. Cis‐regulatory polymorphisms in the OsPDX1.2 promoter fine‐tune its expression and correlate with natural variations in VB6 levels and cold tolerance. Collectively, these findings establish OsPDX1.2 as a key regulator of VB6 synthesis, elucidate the regulatory mechanism underlying VB6 metabolism and stress tolerance, and provide a basis for novel rice breeding strategies to develop vitamin B6‐enriched, stress‐tolerant crops supporting sustainable food security.

We determined that the cold tolerance regulated by OsPDX1.2 depends on the accumulation of VB6 and ABA. Several secondary metabolites, such as proline, branched‐chain amino acids (BCAA), vitamin B5, and VB6, act as effective reactive oxygen species (ROS) scavengers, providing protection against damage induced by stresses, including salt and drought (Mooney and Hellmann, 2010; Kaur and Asthir, 2015; Batista‐Silva et al., 2019; Foyer et al., 2020; Sun et al., 2024). Recent research showed that amino acid metabolism contributes to cold tolerance through a ZmICE1‐dependent mechanism in maize (Jiang et al., 2022). Here, we demonstrate that VB6 enhances rice cold tolerance by scavenging ROS. ABA, an important plant hormone regulating responses to various abiotic stresses, including low temperature, has been shown in numerous studies to significantly enhance cold tolerance in diverse plant species (Baron et al., 2012; An et al., 2022; Parwez et al., 2022; Guan et al., 2023; Zheng et al., 2023; Liu et al., 2024a). VB6 is essential for aldehyde oxidase activity, which catalyzes the final step of ABA biosynthesis (Seo et al., 2000; Bittner et al., 2001; Liu et al., 2022b). Exogenous application of VB6 and ABA rescued the cold stress‐sensitive phenotype of the ospdx1.2 mutant plants (Figure 4). This finding, together with the observed reductions in VB6 and ABA content in ospdx1.2 mutants under cold stress, collectively suggests that OsPDX1.2 regulates cold tolerance through VB6 and ABA synthesis. Additionally, the ospdx1.2 mutant shows suppressed ABA synthesis and altered expression of signaling pathway genes (Figure S12), suggesting that both ABA synthesis and ABA signaling may be involved in OsPDX1.2‐mediated cold tolerance. Nevertheless, further verification is required to establish whether both VB6 and ABA are essential for OsPDX1.2‐mediated cold tolerance.

To our knowledge, no transcription factors that regulate VB6 biosynthesis have been identified in plants. OsbZIP18 is a homolog of AtHY5, a transcription factor known to promote photomorphogenesis and positively regulate cold adaptation and ultraviolet‐B (UV‐B) radiation tolerance in Arabidopsis (Gangappa and Botto, 2016). In tomato, HY5 positively regulates cold tolerance through the C‐repeat binding factor (CBF) signaling pathway (Zhang et al., 2020). Recent research demonstrated that maize HY5 positively regulates cold tolerance in maize by negatively regulating COOL1 expression (Zeng et al., 2025). Our previous results demonstrate that OsbZIP18 positively regulates BCAA and serotonin biosynthesis, while negatively regulating rice tolerance to UV‐B (Sun et al., 2020, 2022; Liu et al., 2024b). We revealed here that OsbZIP18 directly inhibits OsPDX1.2 expression, which in turn suppresses VB6 biosynthesis and reduces cold tolerance. However, knockout of OsbZIP18 did not affect VB6 content or ABA levels in rice. Given the high amino acid sequence similarity between OsbZIP18 and OsbZIP48 (Burman et al., 2018; Xiong et al., 2024), we speculate that these two genes are functionally redundant in regulating cold tolerance. OsbZIP18 and its functional counterpart HY5 in Arabidopsis, tomato, and maize play opposing roles in response to cold and UV‐B stress, indicating evolutionary differentiation in gene function. Further research is, however, required to determine whether HY5 also regulates the synthesis of VB6 in other plant species. A number of genes have been identified that impart cold tolerance in diverse rice subspecies (Saito et al., 2010; Zhang et al., 2017; Li et al., 2021; Lou et al., 2022). Recent research showed that natural variants of OsbZIP73, OsCTF1, OsCTB3, OsCTB5, and OsTCP19 facilitate cold climate adaptation in japonica rice (Liu et al., 2018; Dong et al., 2025; Guo et al., 2025; Li et al., 2025a). Natural variation analyses revealed that pOsPDX1.2 Hap2 conferred higher cold tolerance and enhanced VB6 accumulation compared to pOsPDX1.2 Hap1 . These results define pOsPDX1.2 Hap2 as an elite haplotype that enhances VB6 accumulation and confers cold adaptation in rice. Moreover, we previously identified an elite haplotype of OsbZIP18 associated with reduced transcriptional levels (Sun et al., 2020). The elite variants in OsPDX1.2 and OsbZIP18 may serve as prime targets for gene editing or marker‐assisted breeding to develop rice with both high VB6 content  and stress tolerance.

MATERIALS AND METHODS

Plant materials

The 512 rice accessions used in this investigation are detailed in Dataset S1, including variety names, country of origin, geographical coordinates (longitude and latitude), and subpopulation classifications. The ospdx1.2 mutant was obtained via CRISPR‐Cas9 technology, and the OsPDX1.2‐overexpression line was generated by introducing the OsPDX1.2 coding sequence into the PJC034 vector, followed by Agrobacterium‐mediated transformation (Miao et al., 2013). Both the osbzip18 mutant and the OsbZIP18‐overexpression line were obtained from our previous work (Sun et al., 2022).

Metabolic profiling analysis

Metabolites were extracted and detected as previously described (Shi et al., 2023). Briefly, leaf tissue from 4‐week‐old rice plants and mature seeds was freeze‐dried and then ground into a powder using a mixer mill (MM400, Retsch) at 30 Hz for 1.5 min. 100 mg of the powder was weighed and extracted at 4°C with 70% aqueous methanol containing 0.1 mg/mL lidocaine as an internal standard. After centrifugation and filtration, the extracts were analyzed using LC‐MS/MS (AB SCIEX QTRAP6500). For each transgenic material, at least three independent transgenic lines were selected for targeted metabolite analysis.

Genome‐wide association studies and transcriptome‐wide association studies

Genome‐wide association analysis was performed as previously described (Chen et al., 2014). In brief, genome‐wide SNPs were obtained from the RiceVarMap database. SNPs with a minor allele frequency (MAF) ≥ 0.05 and present in at least six accessions were retained for association analysis. A linear mixed model implemented in the FaST‐LMM program was used to control for population structure by incorporating a kinship matrix as a random effect. The genome‐wide significance thresholds were set at a nominal level of 0.05, corresponding to P = 1.16 × 10−8 for the entire population and to P = 1.78 × 10−8 for the indica subpopulation.

Transcriptome‐wide association analysis was performed as described (Li et al., 2024). Specifically, normalized expression values were transformed to a 0–2 scale. Associations between these transformed expression values and phenotypic measurements were tested using the compressed mixed linear model (CMLM) in the GAPIT software package (version 3.1). A Bonferroni correction for the total number of tested gene expression features was applied to determine statistical significance at α = 0.05.

RNA extraction and expression analyses

Total RNA from rice tissues was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First‐strand cDNA synthesis was performed with 3 μg of RNA using the EasyScript One‐Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen), following the standard protocol. Quantitative real‐time PCR analyses were conducted on an ABI 7500 Real‐Time PCR system (Applied Biosystems) with the SYBR Premix Ex Taq kit (TOLOBIO). Gene expression levels were normalized using the rice UBIQUITIN gene as an internal control. The primers used for these constructs are detailed in Table S5.

Recombinant protein expression and enzyme assay

The full‐length coding sequences of OsPDX1.2 and OsPDX2 were amplified using Nipponbare (NIP) cDNA as a template and recombined into a modified pGEX‐6p‐1 vector (Invitrogen) containing a glutathione S‐transferase (GST) tag. The recombinant vectors were transformed into E. coli strain BL21 (DE3), and protein expression was induced by adding 0.1 mM isopropyl‐β‐D‐thiogalactoside (IPTG), followed by protein purification. The presence of target proteins was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and the purified recombinant proteins were selected for an enzyme activity assay. The primers used for these constructs are detailed in Table S5.

The in vitro enzymatic reaction of OsPDX1.2 was conducted in a total volume of 100 μL reaction mixture, which contained 10 mM aldehydo‐D‐ribose 5‐phosphate (R5P), 50 mM D‐glyceraldehyde 3‐phosphate (G3P), 200 mM L‐glutamine (Gln) as substrates, 1 M Tris‐HCl buffer (pH 8.0), 100 mM MgCl2, and 5 μg of purified protein. The reaction was incubated at 37°C in the dark. After 30 min of incubation, 100 μL of ice‐cold methanol was added to terminate the reaction. The reaction mixture was filtered through a 0.22‐μm filter membrane (Anpel) prior to analysis by LC‐MS/MS. The content of pyridoxal‐5‐phosphate (PLP) was determined by peak integration calibrated with standard samples.

Subcellular localization

To determine the subcellular localization of the OsPDX1.2 protein, the full‐length coding sequence of OsPDX1.2 was amplified and the PCR product was cloned into pH7WGF2.0 (PJC032) to generate the 35S:EGFP–OsPDX1.2 construct. This construct, together with the plasma membrane marker CBL1‐OFP, was transiently expressed in N. benthamiana leaves via Agrobacterium tumefaciens‐mediated infiltration, and was also maxi‐prepared and transformed into protoplasts isolated from ZH11 rice seedlings. Two d after transformation (for tobacco) or after overnight incubation (for protoplasts), fluorescence signals were observed under a confocal laser scanning microscope (Zeiss, LSM980). The primers used for these constructs are detailed in Table S5.

Transient dual‐luciferase reporter assay

To examine the activity of OsPDX1.2 promoter haplotypes, the OsPDX1.2 promoter sequences from NIP (japonica) and ZS97 (indica) were amplified and cloned into a modified pH2GW7 (PJG077) vector containing both firefly luciferase (LUC) and Renilla luciferase (REN) genes. These plasmids were introduced into A. tumefaciens strain EHA105 and infiltrated into N. benthamiana leaves. Luciferase activities were measured using the Dual‐Luciferase Reporter Assay System (Promega) following the manufacturer's instructions.

For investigating the regulatory effect of OsbZIP18 on OsPDX1.2, site‐directed mutagenesis (vg1000082732, G‐C) was performed using the aforementioned amplified NIP promoter as a template, and the resulting sequence was ligated into the PJG077 vector. The full‐length coding sequence of OsbZIP18 was cloned into the pEAQ HT‐DEST2 (PJF754) vector to serve as the effector. Measurements were conducted using the same method as described above, and luciferase activity was calculated as the ratio of LUC to REN (LUC/REN). The primers used for these constructs are detailed in Table S5.

Yeast one‐hybrid

The promoter sequence of OsPDX1.2 was amplified and ligated into the pHIS2 reporter vector. The full‐length cDNA sequence of OsbZIP18 was amplified and fused with the pGADT7 vector. These fusion vectors were then co‐transformed into Y187 yeast cells (Clontech) and cultured on SD/–Leu–Trp medium for 3 d, followed by transfer to SD/–Leu–Trp–His medium for screening. The corresponding combination of empty pGADT7 with the pHIS2‐OsPDX1.2 was co‐transformed as a negative control.

Electrophoretic mobility shift assay

The full‐length cDNA of OsbZIP18 was cloned into the pGEX‐6p‐1 vector with a GST tag. This recombinant plasmid was then transformed into E. coli strain BL21 (DE3) cells for protein expression, and the recombinant OsbZIP18 protein was obtained following purification. FAM‐labeled probes containing the ACE motif were synthesized for electrophoretic mobility shift assay (EMSA). Following this, reaction mixtures were prepared and incubated as previously described (Xian et al., 2025). The relevant primer and probe sequences are listed in Table S5.

Exogenous application of VB6 and ABA

All seeds were germinated on distilled water‐saturated filter paper at 37°C for 3 d. After germination, the seeds were transferred to plastic containers for hydroponic culture in a climate chamber, with growth conditions maintained as follows: white light intensity of 120 μmol m−2 s−1, photoperiod of 16 h light/8 h dark, and temperature of 25°C. A dose–response analysis determined that 30 μM PN (VB6) and 30 μM ABA were the optimal concentrations for exogenous application. Twelve‐d‐old seedlings were subjected to cold treatment at 4°C for 5 d, followed by a 7‐d recovery period under 25°C conditions. For VB6 and ABA treatments, 30 μM solutions of pyridoxine and ABA were sprayed onto the leaves of rice seedlings prior to the initiation of cold treatment. Spray applications continued daily throughout the 5‐d cold treatment period, while control seedlings were sprayed with an equal amount of water. Plants were cultivated using a basal nutrient solution as described previously (Yoshida et al., 1976). The nutrient solution was renewed every 3 d.

DAB and NBT staining

The diaminobenzidine (DAB; Sangon Biotech) and nitrotetrazolium blue chloride (NBT; Sangon Biotech) staining assays were performed following the method as previously described, with minor modifications (Liu et al., 2025b). Following a 24‐h treatment at 4°C, leaf samples were immediately collected and immersed in centrifuge tubes containing either 0.05% DAB (w/v) or 0.1% NBT (w/v) staining solution. The samples were then subjected to vacuum infiltration for 30 min and subsequently incubated in the respective staining solutions for 12 h (DAB) or 8 h (NBT) in the dark. Finally, the stained leaves were placed in 95% ethanol and incubated at 80°C until the decolorizing solution turned colorless.

Quantification of malondialdehyde contents

The malondialdehyde (MDA) content in rice leaves was determined using the MDA content assay kit (D799761‐0050; Sangon Biotech). Approximately 0.1 g of leaf tissue was homogenized in 1 mL of ice‐cold extraction buffer. The homogenate was then centrifuged at 4°C and 8,000 g for 10 min. The supernatant was collected, and the MDA detection working solution was added to it. The absorbance values were measured at 532 nm and 600 nm, and these readings were used to calculate the MDA content.

Statistical analysis

Data were analyzed using GraphPad Prism 8.0 and Microsoft Office Excel. Statistical significance between groups was determined by one‐way ANOVA (Tukey's test), the Wilcoxon rank‐sum test, and a two‐tailed Student's t‐test, as appropriate. Results are expressed as means ± SE, and exact P‐values are included in the figure legends.

Accession numbers

Sequence data used in this study can be found in the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) under the following accession numbers: OsPDX1.2 (LOC_Os10g01080), OsPDX2 (LOC_Os02g03740), and OsbZIP18 (LOC_Os02g10860). The relative contents of pyridoxine (PN) and pyridoxine‑glucoside (PN‑G) for the 512 rice accessions are provided in Dataset S1.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Y.Z. and C.J. conceived and designed the study. B.W., Q.W., and W.S. performed the majority of experiments; R.L., X.Z., L.W., S.X., K.C., J.Z., Y.S., J.H., and X.L. assisted with experiments. Y.Z. and C.J. wrote and finalized the manuscript; A.F., Y.H., J.L., and J.L. discussed the results and revised the paper, and C.J. finalized the paper. All authors have read and approved the contents of this paper.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70322/suppinfo

Dataset S1. Relative contents of pyridoxine (PN) and pyridoxine‐glucoside (PN‐G) in 512 rice (O. sativa L.) varieties

JIPB-68-3914-s003.xlsx (57.8KB, xlsx)

Dataset S2. Rice accessions used for geographical distribution analysis of OsPDX1.2 promoter haplotypes

JIPB-68-3914-s002.xlsx (132.5KB, xlsx)

Figure S1. Chromatographic profiles of vitamin B6 vitamers and their derivatives in the sample

Figure S2. Genetic composition and variation in vitamin B6 metabolite contents across 512 rice accessions

Figure S3. Dissection of a genetic locus underlying natural variations of vitamin B6 in rice

Figure S4. Subcellular localization of OsPDX1.2

Figure S5. Metabolic profiling of ospdx1.2 mutants

Figure S6. Metabolic profiling of OsPDX1.2‐overexpressing plants

Figure S7. Expression analysis of OsPDX1.2 under cold stress

Figure S8. Enhanced cold tolerance of OsPDX1.2‐overexpressing plants

Figure S9. ABA content in leaves of ospdx1.2 mutants and OsPDX1.2‐overexpressing plants

Figure S10. Phenotypic analysis of cold tolerance in 36 indica accessions grouped by pOsPDX1.2 haplotypes

Figure S11. Metabolic profiling of osbzip18 mutant plants

Figure S12. Expression levels of ABA biosynthesis‐ and signaling‐related genes in the ospdx1.2 mutant under cold stress

Table S1. Detailed information of the detected metabolites of 7 types of VB6 and their derivatives in this study

Table S2. Summary of candidate genes assigned from mGWAS results

Table S3. Information related to SNPs in OsPDX1.2

Table S4. List of bZIP transcription factors co‐expressed with OsPDX1.2 identified through the ATTED‐II database

Table S5. Primers and probes used in this study

JIPB-68-3914-s001.docx (2.5MB, docx)

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (32260065, U22A20476, 32360068, and 32302664), the Innovation Project of Postgraduates of Hainan Province (Qhyb2023‐86), and the Sanya Science and Technology Innovation Special Project (2022KJCX54).

Biographies

graphic file with name JIPB-68-3914-g003.gif

graphic file with name JIPB-68-3914-g006.gif

Wang, B. , Wang, Q.Y. , Shi, W.J. , Li, R.H. , Zhang, X.D. , Wang, L.P. , Xia, S.J. , Chen, K.X. , Zhou, J.J. , Sun, Y.Y. , et al. (2026). Natural variation in the promoter of OsPDX1.2 enhances vitamin B6 accumulation and cold tolerance in rice. J. Integr. Plant Biol. 68: 3914–3928.

Edited by: Shuhua Yang, China Agricultural University, China

Contributor Information

Yuan Yuan Zhang, Email: yuanyuan.zhang@hainanu.edu.cn.

Jie Luo, Email: jie.luo@hainanu.edu.cn.

Cheng Jin, Email: Jincheng@hainanu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70322/suppinfo

Dataset S1. Relative contents of pyridoxine (PN) and pyridoxine‐glucoside (PN‐G) in 512 rice (O. sativa L.) varieties

JIPB-68-3914-s003.xlsx (57.8KB, xlsx)

Dataset S2. Rice accessions used for geographical distribution analysis of OsPDX1.2 promoter haplotypes

JIPB-68-3914-s002.xlsx (132.5KB, xlsx)

Figure S1. Chromatographic profiles of vitamin B6 vitamers and their derivatives in the sample

Figure S2. Genetic composition and variation in vitamin B6 metabolite contents across 512 rice accessions

Figure S3. Dissection of a genetic locus underlying natural variations of vitamin B6 in rice

Figure S4. Subcellular localization of OsPDX1.2

Figure S5. Metabolic profiling of ospdx1.2 mutants

Figure S6. Metabolic profiling of OsPDX1.2‐overexpressing plants

Figure S7. Expression analysis of OsPDX1.2 under cold stress

Figure S8. Enhanced cold tolerance of OsPDX1.2‐overexpressing plants

Figure S9. ABA content in leaves of ospdx1.2 mutants and OsPDX1.2‐overexpressing plants

Figure S10. Phenotypic analysis of cold tolerance in 36 indica accessions grouped by pOsPDX1.2 haplotypes

Figure S11. Metabolic profiling of osbzip18 mutant plants

Figure S12. Expression levels of ABA biosynthesis‐ and signaling‐related genes in the ospdx1.2 mutant under cold stress

Table S1. Detailed information of the detected metabolites of 7 types of VB6 and their derivatives in this study

Table S2. Summary of candidate genes assigned from mGWAS results

Table S3. Information related to SNPs in OsPDX1.2

Table S4. List of bZIP transcription factors co‐expressed with OsPDX1.2 identified through the ATTED‐II database

Table S5. Primers and probes used in this study

JIPB-68-3914-s001.docx (2.5MB, docx)

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